AU599891B2 - CDNA clones coding for polypeptides exhibiting murine interleukin-2 activity - Google Patents

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Form PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE Short Title:- Int. CI: Application Number, Lodged; Complete Specification-Lodged: Accepted: Lapsed, Published: Priority This document contains the amcridnents ma (Ie under o)f 49 and is correct for Re-lated Art: fNbne of Applicant: towt t c Address of Applicant-, Actual Inventor: Add ress: for Service: TO BE COMPLETED BY APPLICANT SCHERING BIOTECH CORPORATION 1454 Page Mill Road, Palo Alto, CALIFORNIA 94304, U.S,A, Frank Don Lee Takiishi Yokota and Ken-ichi Arai GRIFFITH HASSEL FRAZER 71 YORK STREET SYDNEY, 2 i00, AUJSTRALIA Complete Specification for thc invention entitled: cDNA Clones Coding for Polypeptides Exhibiting Murine Interlteukin-2 Activity The following statement is a full description of this jn'ention, Including the best. method of performinp. it known to me:-* Note, The do, triptlan Is to be typed In double spacing, pica type face, In an area not exceeding 250 mm In depth and 160 mm in width, on tough white paper of good quality and It Is to be Inserted Inside this form, 14599/78- L 145917- LPrinted by C. J. THOMPSON, Commonwealth Government Printer, Canberra d 1

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i :1 cDNA CLONES CODING FOR POLYPEPTIDES EXHIBITING MURINE INTr'RLEUKIN-2 ACTIVITY This invention relates generally to the application of recombinant DNA technology to elucidate the control mechanisms of the mammalian immune response and, more particularly, to the isolation of deoxyribonucleic acid (DNA) clones coding for polypeptides exhibiting murine interleukin-2 activity.

Recombinant DNA technology refers generally to tho technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the g4netic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product., The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.

This technology has progressed extremely rapidly in recent years, and a variety of exogenous proteins have been expressed in a variety of hosts. By IIc ft ft III 4 ft ft i ftC 1 4 I I -2- IA t C t It I t 4 t 4 1 C 44 '444

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L t t 4 44 0 4 44 04 0400 way of example, some of the eukaryotic proteins soproduced include: proinsulin (Naber, S. et al., Gene 21: 95-104 [1983]); interferons (Simon, L. et al., Proc.

Nat,, Acad. Sci. 80: 2059-2062 [1983] and Derynck, R. et al., Nucl. Acids Res. 1: 1819-1837 [1983]); growth hormone (Goeddel, et al., Nature 281: 544-548 [1979]) and mast cell growth factor (Yokota et al., Proc. Nat. Acad. Sci. 81: 1070-1074 [19841. (These publications and other reference materials have been included to provide additional details on the background of the pertinent art and, in particular instances, the practice of invention, and are all incorporated herein by reference.) For some time, it has been dogma that the mammalian immune response was due primarily to a series of complex cellular interactions, called the "immune network". Although it remains clear that much of the response does in fact revolve around the network-like interactions of lymphocytes, macrophages, granulocytes and other cells, immunologists now generally hold the opinion that soluble proteins the so-called lymphokines) play a critical role in controlling these cellular interactions.

Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to have the ability to support the proliferation and growth of various lymphocytes and, indeed, are thought to play a crucial role in the basic differentiation of pluripotential hematopoietic stem cells into the vast number of progenitors of the diverse cellular lineages responsible for the immune response. Cell lineages important in this response include two classes of lymphocytes: B cells, which can produce and secrete immunoglobulins (proteins with the capability of r ~111--

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it -3recognizing and binding to foreign matter to effect its removal); and T cells (of various subsets) that induce or suppress B cells and some of the other cells (including other T cells) making up the immune network.

Research to better understand (and thus potentially treat therapeutically) immune disorders, through the study of B cells, T cells and the other cells involved in the immune response, has been hampered by the general inability to maintain these cells in vitro, such cells could be isolated and cultured by growing them on secretions from other cells, conditioned media from splenic lymphocytes stimulated with Concanavalin A (ConA). It has now become clear from this work that the generation of cell clones is dependent on specific factors, such as lymphokines.

Some of the better characterized lymphokines are the so-called interleukins, lymphocyte activating factor (LAF), which is released from macrophages and can induce replication of thymocytes and peripheral T cells (Mizel, S. et al., J. Immunol. 120: 1497-1503 [1978]), T cell growth factor (TCGF), which was initially detected in conditioned media from lectinstimulated lymphocytes (Morgan, D. et al., Science 193: 1007-1008 [1976]), and mast cell growth factor (MCGF), which was found in lectin-stimulated T cell clones (Nabel, G. et al., Nature 291: 332-334 [1981]). In 1979, the Second International Lymphokine Workshop labelled LAF as interleukin-1 and TCGF as interleukin-2 Similarly, although not officially, MCGF is now known generally as interleukin-3 (IL-3) (Ihle, J. et al., J. Immunol. 131: 282-287 [1983]).

In view of the central role T cells play in the immune response, their growth factor, IL-2, has been the I 44 4 4, 4r 1 4 t 4 4 4 4OIC 0~

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ii i' i r ;r~j t rr oti r *o 1 84 8 8*4 44 8 84 .444 ro 8 -4subject of considerable study since its discovery about years ago (Smith, K. Immunol. Rev. 51: 337-357 [1980]). IL-2's prime function is almost certainly the stimulation and maintenance of proliferation of most T cell subsets. In fact, the removal of IL-2 from proliferating T cells results in their death within a few hours (Ruscette, F. et al., J. Immunol. 123: 2928-2931 [1977]). IL-2 has also been shown to be one of the lymphokines responsible for cytotoxic T lymphocyte generation, as well as differentiation and induction of function.

Importantly, IL-2's function is not restricted to being just a growth factor for activated T cells. The secretion by T cells of y-interferon and B cell growth factors appears to be induced by IL-2 (Torres, B. et al., J. Immunol. 126: 1120-1134 [1982] and Howard, M. et al., J. Exp. Med. 158: 2024-2039 [1983]). Indeed IL-2 may be at the center of the lymphokine-mediated immune response (for a detailed description of IL-2 activities, see Farrar, J. et al., Immunol. Rev. 63: 129-166 [1982]).

Several procedures have been developed for the isolation and purification of IL-2 Watson, J. et al., J. Exp. Med. 150: 849-855 [1979]; Engleman, E. et al., J. Immunol. 127: 2124-2127 [1981]; and U.S. Patents Nos. 4,404,280 and 4,407,945). These procedures generally entail culturing normal spleen cells or certain lymphoma cell lines, and then stimulating the cells with a mitogen. This often results in relatively low concentrations of IL-2 in the cell media, so that subsequent concentration and purification presents a formidable task.

Furthermore, the IL-2 preparations almost undoubtedly contain some residual mitogen, as well as contaminating proteins from the IL-2-producing cell r 1 j ':7,r *i i i 45 4 4 4 *4 *444 4i *i *4 4 50 44 5 4 .444o 4r~ line. The development of murine T cell hybridomas producing IL-2 has mitigated the mitogen contamination, but the problem remains that most, if not all, murine IL- 2 preparations contain other immunological proteins.

These proteins can influence assay results, and thus interfere with the unequivocable determination of the precise range of IL-2 activities.

In the human system, these problems have been basically alleviated by the successful cloning and expression of cDNAs encoding for human IL-2 (Taniguchi, T. et al., Nature 302: 305-310 [1983] and Devos, R. et al., Nucl. Acids Res. 11: 4307-4323 [1983]), but the problems remain in the murine system. Given that most i;munological experiments are still performed on mice or mouse-derived cells, research is still greatly hampered.

Indeed, the molecular properties of IL-2 remain uncertain. Although it presently appears that the molecular weight of IL-2 is approximately 30-35,000 daltons (Shaw, J. et al., J. Immunol. 120: 1967-1973 [1978]), at least one investigator believes that murine IL-2 is a dimer made up of two 16,000 dalton components (Caplan, B. et al., J. Immunol. 126: 1351-1354 [1981]). Translation in Xenopus laevis oocytes of sizefractionated mRNA (indicating that one or more murine mRNA species of about 1000 to 1100 nucleotides encode a protein exhibiting IL-2 activity) has shed some light on the question (Bleackley, R. et al., J. Immunol. 127: 2432-2435 [1981]), but research has still been slowed by the absence of coding sequences and means for producing large quantities of the desired protein.

Clarification of many of the outstanding issues relating to the molecular biology of murine interleukin-2 requires cdditional structural data, substantially full-length sequence -analysis of the protein and nucleic .y--aa -6acid molecules in question. Protein sequencing offers, of course, a possible means to resolve the matter to a certain degree, but it is very difficult work experimentally and often can provide neither completely accurate nor full-length amino acid sequences. In fact, murine IL-2 appears to be blocked near the NH2 terminus, rendering protein sequencing even more difficult.

Moreover, having the capability of making bulk quantities of a polypeptide exhibiting murine IL-2 activity (and substantially free from other murine proteins) will greatly facilitate the study of the biology of T cells and other cells involved in the immune response; by minimizing the necessity of relying on lectin-conditioned media for stimulating cell growth.

Accurate and complete sequence data on murine IL-2 will help elucidate the structure of that compound and also Sserve to simplify the search for other immunological S, factors. Finally, additional information on any Slymphokine will help in evaluating the roles of the t' various growth factors and cells of the immune network t| and thus provide insight into the entire immune system with the concomitant therapeutic benefits.

Thus, there exists a significant need for extensive nucleotide sequence data on the DNAs coding for, and for the amino acid sequences of, proteins exhibiting murine IL-2 activity, as well as a simple and j economic method of making substantial and essentially S! pure quantities of such materials. The present invention I fulfills these needs.

The present invention provides cDNA clones coding for polypeptides exhibiting murine IL-2 activity. The cDNA sequence can be integrated into various vectors, which in turn can direct the synthesis of the corresponding polypeptides in a variety of hosts, 1.

-7including eukaryotic cells, such as maunalian cells in culture.

The invention also provides a polypeptide exhibiting murine and having the struicture: a process for pro(' -ing interieukin-2 activity

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Met Leu Thr Gin Met Arg Tyr Cys Asp Giu Leu Asp Ile Tyr Val1 S er Gin Asp Asn Leu Leu Leu Asn Lys Giu Ala Ser Leu Ser Gin Leu Leu Pro G iu Thr Ph e GJ y Ser Phe Met Gin Leu Val Ser Thr Gin Gin Gln Giu Lys Leu Lys Gin Asp Giu Gin Ser Ile Ser Ser Asp Ala Thr Cys Gin Leu Asn Ala Gin Leu Pro Ala Leu Lys Asn Asn Val1 Ser Ala Ser GiU Gin Leu Arg Thr Gly Ser Ile Thr Val Ile Ser Ala Ala His Ser Met Giu Pro Phe Arg Phe Asp Ile Cys Pro Gin Leu Arg Leu.

Leu Leu Gin Val Giu Ph e Ser Val Thr Gin G iu Met Thr Lys Arg Leu Thr Cys Leu Thr Thr Ser Gin Gin Giu.

Phe Asp His G 11- Vali G In Arg Ser Leu S e'r Gin Leu Asn Lys Leu Val Asp Val1 Phe Arg Pro Thr Ser Gin Leu Tyr Phe Gin Leu Ala Ly s Asp Trp Gin, 5456

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SI I S J S 5515 said process comnprising the steps of: a) Providing a vector comprising a nucleotide sequence coding for said polypeptide, wherein tlie nucleotide sequence is capable of being expressed by a host containing the vector and is a cDNA sequence derived from an TnRNAL sequ~ence coding for said polypeptide; h) incorporating the vector into the host; and c) maintaining the host containing the vector under conditions suitable for expression of the nucleotide sequence into said polypeptide.

-7A- Preferably, the host is an organism such as a eukaryotic, e.g. mammalian, cell transfected or transformed with the vector. Further, the vector preferably comprises also a second nucleotide sequence capable of controlling expression of the nucleotide sequence coding for the polypeptide. This second sequence coding can include a promoter sequence, one or more intron sequences and a polyadenylation sequence, to permit, respectively, transcription, splicing and polyadenylation of the nucleotide sequence coding for the polypeptide. Particularly, when the host is a mammalian cell, such as a COS-7 (monkey kidney) cell, the vector contains the promoter sequence of the simian virus 40 early region promoter and the polyadenylation sequence of the SV40 late region polyadenylation sequence.

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444, -8- The mouse cDNA sequence, or a portion thereof, is capable of hybridizing with other DNA sequences, such as DNA coding for other mammalian growth factors from a cDNA or genomic libr'ary. It is noted that the described cDNA sequences seem to contain infr mation for a leader sequence.

The polypeptides of the present invention are capable of enhancing growth of murine T cell and other cells, particularly in cultures in vitro. Suitable compositions for i'ustaining IL-2 receptive cell lines can be prepared by adding the polypeptides (preparations of which are essentially free of other murine growth factors) to well known cell growth media formulations, Other features and advantages of the inventi.on will become apparent from the following detailed description, which describes, in conjunction with the accompanying drawings and by way of example, the present invention.

Figure 1 illustrates the nucleotide sequence of a cDNA clone and putative corresponding amino acid sequence of a polypeptide exhibiting murine IL-2 activity; Figure 2A illustrates pcD-IL-2, a plasmid carrying a cDNA clone exhibiting IL-2 activity; Figure 2B is a restriction endonuclease cleavage map of the cDNA insert of Figure 2A.

Figure 3 represents a comparison between putative murine and human IL-2 amino acid sequences.

Figure 4A shows the structure of yeast preproalpha-factor, including the amino acid sequence of the first spacer alpha-factor and a portion of the second spacer.

Figure 4B shows the construction of a general yeast construction vector, designated pMFa8.

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it Ii _I1Cli 1 I -9- In accordance with the present invention, cDNA clones are provided that code for polypeptides exhibiting IL-2 activity. After the cDNA sequences have been incorporated into replicable expression vectors, and the vectors transfected into an appropriate host a mammalian cell culture), the expressed polypeptide or polypeptides have the ability to allow the expansion of T cells and their related lineages.

An exemplary, putative amino acid sequence based on the isolated nucleotide sequence is shown in Figure 1. The murine IL-2 cDNA contains a single openreading frame consisting.of 168 codons. Downstream of the putative initiation codon is a region rich in hydrophobic amino acids. Including the asparagine codon at amino acid position 19, there is a stretch of nine continuous amino acids identical to that predicted for human IL-2. The alanine res' lue in this sequence coincides with the NH -terminal amino acid deduced for human IL-2 (Stern, A. et al., Proc. Nat. Acad. Sci.

o U.S,A. 81: 871-875[1984]). It is likely, therefore, S* that the mature form of secreted mouse IL-2 begins with 0 Go an alanine residue, and the preceeding 20 amino acids constitute a leader region, which is subject to removal by proteolytic processing. Thus, mature murine IL-2 would consist of about 149 amino acids, with a calculated molecular weight of approximately 16,000 daltons (unglycosylated). The mature protein probably contains acidic (Asp Glu) and 14 basic (Arg Lys) amino *acids, the difference in numbers perhaps accounting for the acidic isolectric point (pI= 4 3 4 of the protein (Watson, J. et al., J. Exp. Med. 150: 849-861 [1979]).

There do not appear to be any potential N-glycosylation sites (Asn-X-Ser/Thr) (Neuberger, A. et al., Glycoproteins 5, 450-490, Elsevier Publishing Co., U.S.A.

h. f [1972]), however, the presence of other in vivo modifications, such as sialylati'on or 0-glycosylation, cannot be excluded.

When transfected into COS-7 monkey cells or other suitable expression systems, cDNA clones of this invention can direct the synthesis of biologically active murine IL-2. Addition of this exprtomed cloned gene product to cultures of mouse T cells allows expansion of receptive cells and/or their maintenance in culture. The expressed polypeptides can exhibit the assorted activities associated with murine IL-2.

A variety of methods may be used to prepare the cDNAs of the present invention. By way of example, total mRNA is extracted as reported by Berger, S. et al., Biochemistry 18: 5143-5149 [1979]) from cells (eg.

a hybrid cell line) producing polypeptides exhibiting murine IL-2 activity. The double-stranded cDNAs from this total mRNA can be constructed by using primer- S initiated reverse transcription (Verma, Biochim.

Biophys. Acta, 473: 1-38 [19771) to make first the complement of each mRNA sequence, and then by priming for j *second strand synthesis (Land, H. et al., Nucleic Acids Res., 9: 2251-2266 [1981]). Subsequently, the cDNAs can be cloned by joining them to suitable plasmid or bacteriophage vectors (Rougeon, F. et al., Nucleic Acids 1• Res., 2, 2369-2,378 [1975] or Scherer, G. et al., Dev.

Biol. 86, 438-447 [19811) through complementary homopolymeric tails (Zfstratiadis, A. et al., Cell, *571-585 [1977]) or ,ohesive ends created with linker segments containing appropriate restriction sites (Seeburg, P. et al., Nature, 270, 486-494 [1977] or Shine, J. et al., Nature, 270, 494-499 [1977]), and then transforming a suitable host. (See generally Efstratiadis, and Villa-Kormaroff, "Cloning of 1 -11double stranded cDNA" in Setlow, J. and Hollaender, A.

(eds.) Genetic Engineering, Vol. 1, Plenum Publishing Corp., U.S.A. [1982].) A preferred method of obtaining the full-length cloned cDNAs of this invention is the procedure developed by H. Okayama and P. Berg (Mol. and Cell, Biol., 2: 161- 170 [1982]). This method has the advantage of placing the cDNA inserts in a bacterial cloning vector at a position whereby the cDNA can also be directly translated and processed in mammalian cells. Briefly, the first cDNA strand is primed by polydeoxythymidylic acid covalently joined to one end of a linear plasmid vector DNA. The plasmid vector is then cyclized with a linker DNA segment that bridges one end of the plasmid to the end of the cDNA coding sequence. By employing a DNA fragment containing the Simian Virus 40 (SV40) early region promoter and a linker containing a modified late region intron, the cDNA can be expressed in vitro in COS-7 mouse (kidney) cells without further modification. (See generally Okayama, H. and Berg, P., S Mol. and Cell. Biol., 3: 280-289 [19831 -nd Jolly, D. et al., Proc. Nat. Acad. Sci. 80: 477'451 [1983].) ,o Once the cDNA library in the Okay ta/Berg plasmid vector has been completed, the cDNA clones are collected, and random pools are checked for the presence of the desired cDNAs by hybiid selection, translation, and assay by measuring murine IL-2 activity, the J existence of antigenic determinants, or other biological activities). Pools positive by these criteria can then .be probed with an appropriate subtracted probe, e.g., cDNA from a B cell line and/or uninduced T cell line.

Thereafter, the positive, probed pools are divided into individual, clons which are tested by transfection into a suitable host (such as a mammalian cell culture), and the I I -12host supernatant assayed for the desired activity (e.g.

TCGF activity). Positive clones are then sequenced.

The desired cDNA clones can be detected and isolated by hybridization screening with appropriate mRNA samples (Heindell, H. et al., Cell, 15: 43-54 [1978]).

Alternatively, the cDNA libraries can be screened by hybrid selection (Harpold, M. et al., Nucleic Acid Res., 2039-2053 [1978] or Parnes, J. et al., Proc. Nat.

Acad. Sci. 78: 2253-2257 [1981]) or in Xenopus oocytes (Aurdon, Nature, 233: 177-182 [1971]). (See generally Villa-Komaroff, L. et al., Proc. Nat. Acad.

Sci. 75: 3727-3731 [1978].) In further describing the procedures relating to preparing cDNA clones of the invention, the assay cell line and other lines vill be considered first, followed by general descriptions of the procedures for isolating mRNA coding for a protein exhibiting murine IL-2 activity; the construction of a cDNA library containing the cDNA sequences; isolation of full-length cDNA clones in a plasmid vector and subsequent expression in mammalian cells; subcloning and expression in bacteria and yeast; and purification and formulation. A more detailed description of the entire experimental process will follow thereafter.

T Cell and Other Lines Any of a large variety of different cells may be used as sources for murine IL-2 activity and in the assay thereof (see, Robert-Guroff, M. et al., "T-Cell Growth Factor"; Growth and Maturation Factors, ed. Guroff, New York: John Wiley and Sons, pgs. 267- 308 at 287 [1984] and U.S. Patent Nos. 4,404,280 and 4,407,945). A preferred source is LB2-1 (ATCC accession number CRL-8629), but the mouse T lymphoma EL-4 (Farrar,

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464r 4 4 4 4 94 .4 4 4 44 4, 1 i u 1 -13- J. et al., J. Immunol. 125: 2555-2558 [1980]) or other cell producing IL-2 activity is acceptable. The LB2-1 cell line was derived from a C57BL/6 mouse immunized with chicken red blood cells (CRBC) of the MHC genotype

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2 This line can be stimulated by B 2

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2 CRBC in the presence of syngeneic spleen cells, by allogeneic spleen cells bearing the a or d m/s haplotype, or by ConA. LB2- 1 can be grown on alternating cycles of antigen plus spleen stimulator cells followed by several days of IL-2dependent proliferation (see, Clayberger, C. et al., J.

Exp. Med. 157: 1906-1919 [1983]).

Similarly, preferred cell lines for use in connection with murine IL-2 activity assays are the HT-2 line and those developed as described by Watson, J. (J.

Exp. Med. 157: 1906-1919 [1983]). Mouse thymocytes (Disabato, G. et al., Cell. Immunol. 17: 494-504 i| [1975]), cloned cytotoxic T cell lines (Morgan, D. et al., Science 193: 1007-1008 [1976]), and other murine SIL-2 responsive lines are also suitable. The HT-2 line t may be grown in complete medium, which includes RPMI 1640, 10% fetal bovine serum, 0.05 mM 2-mercaptoethanol (2-ME) and partially purified spleen cell supernatant.

To determine IL-2 activity using the HT-2 cells, a colorimetric proliferation assay is preferably utilized (Mosmann, J. Immunol. Methods 65: 55-63 I However, the microassay procedure of Gillis, S.

j et al. of Immunol. 120: 2027-2032 [1978]), indirect Sassays Granelli-Pipeno, A. et al., J. Exp. Med.

B 154: 422-430 [1981]), or other well known assay systems S- are suitable. Units of TCGF can be calculated by S determining the dilution of factor required to give of the maximum stimulation, with one unit defined as the amount of IL-2 required to give 50% of the maximum signal using 2,000 HT-2 cells in a volume of 0.01 ml.

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-14- Isolation of mRNA and Construction of a cDNA Library Total cellular mRNA can be isolated by a variety of well-known methods Przybla, A. et al., J. Biol. Chem. 254: 2154-,3158 [1979]), but the preferred is the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (Biochemistry, 18: 5294-5299 [1979]).

If this method is used, approximately 10 ug of polyA+ mRNA, selected on columns of oligo (dT) cellulose, is obtained from 1-2 x 108 activated T cells, such as LB2-1.

The cDNA library from the polyA mRNA can best be constructed using the pcDVl vector-primer and the pLi linker fragment [available from P-L Biochemicals Inc., Milwaukee, WI) according to procedures which result in greatly enriched full-length copies of mRNA transcripts Okayama, H. and Berg, Mol. Cell Biol., 2, 161- 170 [1982] and Mol. Cell Biol., 3, 280-289 [1983]). The plasmid vector, which contains SV40 early promoter and .SV40 RNA processing signals, is designed to promote expression of the cloned cDNA segment in mammalian cells.

S. Using the Okayama and Berg procedure, the cycoized vector-cDNA preparation is transformed into a competent bacterial cell, such as E. coli MC1061 cells (Casadaban, M. and Cohen, J. Mol. Biol., 138: 179-207 S, [1980]) using calcium chloride (Cohen, S. et al., Proc.

Nat. Acad. Sci. 69: 2110-2114 [1972]). Starting with 5 pg of polyA+ RNA from ConA-stimulated Cl-Ly 1+2'/9 cells, about 1.5 x 106 independent transformants are 044* 4 obtained. About 104 clones are picked up individually and inoculated into wells of microtiter plates (Flow S.,no Laboratories Inc., McLean, Virginia) containing 200 ul of t"o L-broth, 50 pg/ml of ampicillin, and 7% DMSO. If desired, sublibraries based on the size of cDNA insert are prepared from total cDNA library as described by !t

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Okayama, H. and Berg, P. (Mol. Cell Biol., 3, 280-289 [19831). Briefly, plasmid DNA is digested with SalI, Clal, and HindIII separately, and electrophoresed in 1% agarose gel. After staining with ethidium bromide, the gel is sliced into 7 sections corresponding to cDNA insert sizes of 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, to 6, and more than 6 kilobases DNA is extracted from each slice, recyclized with T4 DNA ligase, and used to transform MC1061. All nucleotide sequencing can be performed according to the procedure of Maxam, A. and Gilbert, W. (Methods Enzymol., 65: 499-560 [1980]).

DNA Transfections into Monkey Cells Approximately 1x10 6 COS-7 monkey kidney cells are seeded onto 60 mm plates the day prior to transfection. Transfections are best performed with ig of plasmid DNA in 1.5 ml of DME containing 50 mM Tris.HCl, pH 7.4, and 400 g,/ml DEAE-Dextran (Pharmacia Fine Chemicals, Uppsala, Sweden). This solution is then removed after 4 hr and replaced with 2.0 ml DME 4% fetal calf serum. The medium is collected after 72 ir and assayed for murine IL-2 activity as described above. DNA transfections may be carried out in L-cells and a variety of other cell sources as well (see below).

Expression in E. coli, in Yeast and in Cell Culture Prokaryotes, such as E. coli, are very suitable for expression of the polypeptides of the present invention (see, for example, U.S. patent numbers 4,338,397 and 4,411,994), provided glycosylation is not desired. To obtain high expression levels, promoters should be utilized, such as the P-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature, 275: 615 [1978]; Itakura et al., Science,

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-16- 198: 1056 [1977]; Goeddel et al., Nature 281: 544 [1979] or a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res., 8: 4057 [19801) in conjunction with Shine-Delgarno sequences.

Those skilled in the art will realize that not only prokaryotes but also eukaryotic microbes, such as yeast, may also be used in protein production.

Saccharomyces cerevisiae is a preferred eukaryotic microorganism. Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 12073- 12080 [1980]) or other glycolytic enzymes (Hess et al., Adv. Enzyme Reg., 7: 149-167 [1969]; Holland et al., Biochemistry, 17: 4900-4907 [1978]). Other promoters that have the additional advantage of transcription controlled by growth conditions may be used. Basically any plasmid vector containing a yeast-compatible promoter, an origin of replication and termination tsequences is suitable.

The preferred method of making murine IL-2 I employing the cDNAs of the present invention utilizes the yeast mating pheromone a-factor secretory pathways ,r (Julius, D. et al., Cell 32: 839-852 [1983). S.

cerevisiae secretes mating-type specific oligopeptide pheromones. MATa cells secrete a-factor, which induces *the growth arrest of MATa cells at G1 phase of the cell cycle (Thorner, "The Molecular Biology of the Yeast SSaccharomyces", Cold Spring Harbor Laboratory, NY [1981]; ssee particularly pages 143-180). The a-factor is S initially synthesized as a larger precursor molecule 1 consisting of an NH 2 -terminal signal sequence of about amino acids, followed by an additional 60 amino acids leader sequence and ending with four identical tandem repeats of the mature a-factor sequence. The repeats are i I r -17separated from each other by six or eight amino acids spacers (Lys-Arg-Glu-Ala-Glu-Ala and Lys-Arg-Glu-Ala-Glu- [or Asp-]-Ala-Glu-Ala). This prepro-a-factor is cleaved at several specific sites. The first processing is the cleavage of the COOH-terminal side of the Lys-Arg pair of the spacer sequence catalysed by the KEX2 product (Julius et al., Cell 37: 1075-1089 [1984]). Carboxypeptidase-B like enzyme cleaves at the NH 2 -terminal side of the Lys- Arg pair. The final step is the removal of Glu-Ala or Asp-Ala pairs by diaminopeptidase, which is encoded by the STE13 (See Figure 4A). Brake, J. et al. (Proc. Nat.

Acad. Sci. U.S.A. 81: 4642-4646 [1984]) have shown that the fusion of the sequence encoding mature human proteins (including human IL-2) to the first processing site allowed secretion of such proteins.

A general yeast expression vector, designated pMF-alpha-8, containing the alpha factor promoter and downstream leader sequence in conjunction with other I ,elements, has been deposited with the ATCC (accession Snumber 40140). It can be constructed as follows (See Figure 4B): A 1.7 kb EcoRI fragment carrying the MFal gene (Kurjan, J. and Hershowitz, Cell. 30: 933-943 [1982]) is cloned into the EcoR' restriction site of M13mp8 (Viera, J. and Messing, Gene 19: 259-268 In order to introduce a HindIII site after the lysine codon of the first spacer region, the synthetic I' oligonucleotide TCTTTTATCCAAAGATACCC is hybridized to the single strand M13-MFal DNA and the oligonucleotide primer Sextended by DNA polymerase I Klenow fragment. After Sl nuclease treatment, the DNA is cleaved with EcoRI, and Sthe fragment carrying the MFal promoter and leader sequence is cloned into the EcoRI and filled-in HindIII restriction sites of pUC8 (Viera, J. and Messing, J.

I -18above). One plasmid with the desired structure can be isolated (designated pMFa4A1 in Figure 4B). The pMFa4AI is cleaved with HindIII and partially filled in with DNA polymerase I Klenow fragment in the presence of dATP and dGTP. The DNA is treated with mung bean nuclease, and the oligonucleotide linker GCCTCGAGGC is attached. The resultant plasmi.d (designated pMFa5 in Figure 4B) will have a StuI cleavage site immediately after the arginine codon, followed by the Xhol restriction site. An S.cerevisiae-E.coli shuttle vector (pTRP584) can be constructed as follows: the PstI-XbaI fragment carrying 2 um plasmid replication origin (Broach, J. above) is cloned into the Clal restruction site of pTRP56 (Miyajima et al., Mol. Cell. Biol. 4: 407-414 [1984]), and the StuI restriction site within the TRPl-ARS1 fragment is converted into a PvuII restriction site by PvuII linker insertion. The KpnI restriction site in the original pTRP56 is converted to XhoI by the XhoI linker insertion. The general secretion vector pMFa8 is then t obtained by insertion of the BglII-XhoI fragment of into the BamHl-XhoI restriction sites of pTRP584.

t Those skilled in the art will realize that cDNA tu clones encoding for murine IL-2 may then be readily inserted into the pMFa8 vector and subsequently transformed in yeast for IL-2 production. By way of i example, the PstI-BamHl fragment carrying the entire IL-2 i i cDNA is transfered from pcD-IL-2 into the PstI-BamHl J sites of pUC9 (Viera, J. and Messing, J. above) and i cleaved with HinPl and SmaI. Smal cleaves downstream of the cDNA insert. The fragment is treated with DNA polymerase I Klenow fragment in the presence of dCTP and o, |treated with mung bean nuclease. The fragment is then cloned into the StuI restriction site of pMFa8. This plasmid DNA (carrying the TRP1 gene) can be introduced i -19into yeast cells by the lithium acetate method (Ito, H.

et al., J. Bacteriol. 153: 163-168 [1983]) and transformants are selected in synthetic medium lacking tryptophan. Transformants are then grown in a common medium supplemented with 0.5% casamino acids. For harvesting, the yeast cells, resuspended in phosphatebuffered-saline (PBS) containing 1 mM PMSF, are disintegrated by vigorous shaking with acid washed glass beads. Clear supernatant is obtained by centrifugation at 10,000 rpm for 15 min.

In addition to microorganisms, cell cultures derived from multicellular organisms (especially mammalian cells) may also be used as hosts. Examples of such useful host cell lines are HeLa cells, Chinese hamster ovary cell lines, and baby hamster kidney cell lines. Expression vectors for such cells ordinarily include, as necessary, an origin of replication, a promoter located in front of the gene to be expressed, along with any required ribosome binding sites, RNA splice sites, polyadenylation sitss, and transcriptional S, terminator sequences. When used in mammalian cells, the Sexpression vector often has control functions provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently SV-40. (See, U.S. patent No. 4,399,216 and d Gheysen, D. and Fiers, J. of Mol. and Appl. Genetics 1: 385-394 [1982].) '"Purification and Formulations The murine IL-2 polypeptides expressed in E.coli, in yeast or in other cells can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fractionation column chromatography ion exchange, gel filtration, i electrophoresis, affinity chromatography, etc.) and ultimately crystallization (see generally "Enzyme purification and Related Techniques", Methods in Enzymology, 22: 233-577 [1977]). Once purified, partially or to homogeneity, the polypeptides of the invention may be used for research purposes, as a supplement to cell growth media minimum essential medium Eagle, Iscove's modified Dulbecco Medium or RPMI 1640; available from Sigma Chemical Company, St. Louis, MO and GIBCO Division, Chagrin Falls, OH) and as an antigenic substance for eliciting specific immunoglobulins useful in immunoassays, immunofluorescent stainings, etc. (See generally "Immunological Methods", Vols. I II, Eds. Lefkovits, I. and Pernis, Academic Press, New York, N.Y. [1979 1981]; and "Handbook of Experimental Immunology", ed. Weir, Blackwell U Scientific Publications, St. Louis, MO [1978].) If yeast cultures containing cDNAs of the present invention are utilized to prepare murine IL-2, purification preferably is accomplished as follows. Up to one liter of 12 hr culture fluid is diluted with an ji equal volume of 0.2% trifluoroacetic acid (TFA) and

CH

3 CN, and pumped directly onto a 4.6 X 75 mm propyl column (Ultrapare RPSC, Altex, Berkeley, CA) previously equilibrated with 0.1% TFA, 20% CH 3 CN. Sample r application can be at a flow rate of about 1 ml/min at 4 4°C. When all the sample has been applied, the column is *94 connected to a conventional HPLC system at room 9 temperature, and further washed at 0.5 ml/min until the absorbance monitored at 214 nM reaches baseline. A 041f gradient of increasing solvent concentration is then applied CH 3 CN per min), and 1 ml fractions collected. The murine IL-2 elutes about, 65 min after the start of the gradient in a peak well seyarated from other -21proteins. Analysis of the peak fractions on silverstained Laemmli gels (Laemmli, Nature 227: 680-685 [19701) should indicate that the protein is >95% pure.

The following experimental information and data are offered by way of example and not by way of limitation.

EXPERIMENTAL

A. Cloned LB2-1 T Cells 1) A clone of T Cells LB2-1 (ATCC accession number CRL-8629) expressing the Thy 1 Ly 1+2 phenotype is continuously maintained at 0.5 x 105 cells/ml in RPMI-1640 with 10% heatinactivated fetal calf serum, 5 x 10 5 M 2-ME, 2 mM glutamine, non-essential amino acids, and essential vitamins conditioned with 5% DEAE g* treated supernatants from ConA treated Lewis strain rat spleen cells.

S. 2) ConA-activation of LB2-1 cells: The cells are *cultured at 5 x 10 5 /ml in DME with 4% heatinactivated fetal calf serum, 5 x 10- M 2-ME, 2mM glutamine, non-essential amino acids, S. essential vitamins and 4 ug/ml ConA. After 12- S"lr 14 hrs.' incubation at 37 0 C in 10% CO 2 the cell suspension is centrifuged at 1500 rpm for minutes. The cell pellets are collected and i "frozen immediately at -70 0 C. The supernatants I are filtered (Nalgene-0.22 microns) and stored C at -800C as a source of growth factors.

Aliquots of the supernatant are assayed for murine IL-2 activity (see below) to verify the induction of the line by the ConA treatment.

i -22- B. Cloned HT-2 Cells The T cell line utilized in the murine IL-2 assays was HT-2, a line first described by Watson, J. Exp. Med. 150: 1510-1519 [1979]). It is continuously maintained with doubling times of about 24 hours in RPMI 1640 with 10% heat-inactivated FCS, 5 x 10 5 M 2-ME and 2 mM glutamine, non-essential amino acids and essential vitamins supplemented with supernatant from ConA-activated LB2-1 cells.

The growth of the HT-2 cell clone is dependent on the active growth factor(s) obtained from the supernatant of stimulated LB2-1 cells.

C. Tetrazolium Salt (MTT) Colorimetric Assay for Murine IL-2.

1) About 2000 cells were cultured in flat-bottomed 96 well microtiter trays in 0.1 ml of DME supplemented with co-factors and test supernatant as described above.

U t 2) The trays were incubated at 37 0 C in 10% CO 2 After twenty hours, 0.01 ml of 5 mg/ml MTT (3- S(4,5-dimethylthiazol-2-y tetrazolium bromide, Sigma Chemical Co., St.

i LLouis, MO) in phosphate-buffered saline (PBS) Stt* was added tr each culture. Four hours later J0.1 ml of 0.04 N HC1 in isopropanol was added *ti to each culture and thoroughly mixed. After a few minutes, the plates were read on a Dynatech j MR580 Micr olisa Auto Reader (Dynatech Instruments, Inc., Torrance, CA), at a -23wavelength of 570 nm (reference wavelength of 630nm) and a calibration setting of 1.99.

Ii i it 4 4* 9 4 411* 1111 111 4t .,9 D. Isolation of mRNA from LB2-1 Cells.

1) Total cellular RNA was isolated from cells using the guanidine isothiocyanate procedure of Chirgwin, J. et al., (Biochemistry, 18: 5294- 5299 [1979]).

Frozen cell pellets from uninducod or ConAinduced LBF2-1 cells 8, and 11 hr after stimulation) were suspended in guanidinG isothiocyanate lysis solution. solution was used for 1,5 x 108 cells. Pellets were resuspended by pipetting, and then DNA was sheared by 4 passes through a syringe using a 16 gauge needle. The lysate was layered on top of 20 ml of 5.7 M CsC1, 10 mM EDTA in 40 ml polyallomer centrifuge tube. This solution was centrifuged at 25,000 rpm in a Beckman SW28 rotor (Beckman Instruments, Inc., Palo Alto, CA) for 49 hrs at 150C. The guanidine isothiocyanate phase containing DNA was pipetted off from the top, down to/the interface. The walls of the tube and interface were washed with 2-3 ml of guanidine isothiocyanate lysis solution. The tube was cut below the interface with scissors, and the CsCI solution was decanted. RNA pellets were washed twice with cold 70% ethanol. Pellets were then resuspended in 500 ul of 10 mM Tris.HCl pH 7.4, 1 mM EDTA; 0.05% SDS. S0 p1 of 3M sodium acetate was added and RNA was precipitated with 1 ml ethanolo About 0.3 mg total RNA was collected by centrifuging and the -24pellets washed once with cold ethanol.

2) PolyA mRNA isolation: Washed and dried total RNA pellet was resuspended in 900 pl of oligo (dT) elution buffer (10 mM Tris.HCl, pH 7.4, 1 mM EDTA, SDS). RNA was heated for 3 min. at 68 0 C and then chilled on ice. 100 pl of 5 M NaCI was added. 'he FRNA sample was loaded onto a 1.0 ml oligo (dT) cellulose column (Type 3, Collaborative Research, Waltham, MA) equilibrated with binding buffer (10 mM Tris.HCl pH 7.4, 1 mM EDTA, 0.5 M NaCI, 0.5% SDS.). Flow-through from the column was passed over the column i twice more. The column was then washed with ml binding buffer. PolyA+ mRNA was collected by washing with elution buffer. RNA usually eluted in the first 2 ml of elution buffer.

RNA was precipitated with 0.1 volume 3 M sodium acetate (pH 6) and two volumes of ethanol. The RNA pellet was collected by centrifugation, t washed twice with cold ethanol, and dried. The pellet was then resuspended in water. Aliquots were diluted, and absorbance at 260 nm was i determined.

S

t E. Oocyte Injection Oocytes were removed from female Xenopus laevis and incubated in Barth's solution (88 mM NaCl, 1 mM, i KC1, 0.33 mM Ca(N0 3 2 0.41 mM CaCl2, 0.82 mM MgSO4, 9 2.4 mM NaHC0 3 and 10 mM HEPES (pH (Sigma tj Chemical Co., St. Louis, MO). Injection clusters of 2-3 oocytes were preparod. RNA samples were to be injected dissolved in injection buffer (40 mM Tris.HCl pH 7.4, 0.35 M NaCl) Total pclyA mRNA was resuspended at a concentration of 500 ug/ml in injection buffer, while RNA samples eluted from DNA filters from hybrid selections (see below) always contained 5 jig of calf liver tRNA as carrier and were resuspended in 2 pl of injection buffer. About nl aliquots were injected into each oocyte using micropipets pulled by hand with tips forged using a microforge. The pipettes were calibrated with known volumes of sterile water. Approximately 10 to oocytes were injected for each mRNA sample. The injected oocytes were incubated in groups of two or three in individual wells of 96-well microtiter dishes containing 10 pl of Barth's solution 1% bovine serum albumin per oocyte. The oocytes were kept at 19 0 C for 48 hours. Thereafter, supernatants from triplicate pools of six oocytes each were assayed for IL-2 activity. The supernatants were were first sterilized by centrifuging for 10 minutes it; microcentrifuge and then assayed. Supernatants from uninjected oocytes were always collected as a control.

The assay cesults from supernatants collected from untreated or ConA-stimulated LB2-1 cells are shown in Table I. Titration of all samples, including the reference standard, was performed in triplicate. Units of IL-2 were calculated by determining the dilution of factor requested to give l 50% of maximum stimulation, and one unit is defined as the amount of murine IL-2 required to give 50% of the maximum signal using 2,000 HT-2 cells in a volume of 0.1 ml. L62-1 cells were harvested at 6, 8, and 11 hr following the addition of Con A.

Uninduced cells were grown identically, but omitting -26- Con A. Poly(A)+ RNA was prepared from each cell pellet and then injected into Xenopus oocytes as described. The 8upernatants from stimulated and unstimlate cels and the oocyte supernatants wr 1 assayed for 'ICGF activity on the HT2 cell line.

H

-27- TAB LE I Production of TCGF Activity by LB2-l LB2-1 suroerrmtant 6 cells Oocyte super natant Units/lO6 cell equivalents without Con A with Con A 6 hr 8 hr 11 hr '1 r

I

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10,377 16,496 14,604 44 4 4 4*4 4 4.

4 444 4 44 4' 4 4 4 $4 4 44 4444 4 444 44 4 4 444 t 4 44 4 4 4 4* *411 1 1 414~ 4- 4444*4 4 I #41$

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-28- F. cDNA Library Construction: Preparation of vector primer and oligo dG-tailed linker DNAs: The procedure of Okayama Berg (Mol. Cell.

Biol. 2: 161-170 [1982]) was used with only minor modifications and adapted to the pcDVl and pLl plasmids described by Okayama Berg (Mol. Cell.

Biol. 3: 380-389 [1983]]).

An 80 ug sample of pcDVl DNA was digested at 0 C with 20 U of KpnI endonuclease in a reaction mixture of 450 pl containing 6 mM Tris.HCl (pH 6 mM MgC12, 6 mM NaC1, 6 mM 2-ME, and 0.1 mg of bovine serum albumin (BSA) per ml. After 16 hr the digestion was terminated with 40 pl of 0.25 M EDTA (pH 8.0) and 20 p1 of 10% sodium dodecyl sulfate (SDS); the DNA was recovered after extraction with water-saturated 1:1 phenol-CHC13 (hereafter referred to as phenol-CHCl 3 and ethanol precipitation. HomopQlymer tails averaging 60, but not more than deoxythymidylate (dT) residues per end were added to the KpnI endonuclease-generated termini with calf thymus terminal transferase as follows: The reaction mixture (38 pI) contained souium mM Tris.HC1 pH 6.8 as buffer, with 1 mM CoC1 2 0.1 mM dithiothe itol, 0.25 mM dTTP, the KpnI endonuclease-digested DNA, and 68 U of the terminal deoxynucleotidyl transferase (P-L Biochemicals, Inc., Milwaukee, WI). After 30 min.

at 37 0 C the reaction was stopped with 20 pl of 0.25 M EDTA (pH 8.0) and 10 pl of 10% SDS, and the DNA was recovered after several extractions with phenol-CHCl 3 by ethanol precipitation. The DNA was then digested with 15 U of EcoRI endonuclease 4I [1

CL

t 6 1i *1#1r h 1 -29in 50 ul containing 10 mM Tris.HCl pH 7.4, 10 mM MgC1 2 1 mM dithiothreitol, and 0.1 mg of BSA per ml for 5 hr at 37 0 C. The large fragment, containing the SV40 polyadenylation site and the pBR322 origin of replication and ampicillinresistance gene, was purified by agarose gel electrophoresis and recovered from the gel by a modification of the glass powder method (Vogelstein, B. Gillespie, Proc. Nat, Acad.

Sci. 76: 615-619 [1979]). The dT-tailed DNA was further purified by absorption and elution from an oligo (dA)-cellulose column as follows: The DNA was dissolved in 1 ml of 10 mM Tris.HCl pH 7.3 buffer containing 1 mM EDTA and 1 M NaC1, cooled at 0 C, and applied to an oligo (dA)-cellulose column (0.6 by 2.5 cm) equilibrated with the same buffer at 0°C. The column was washed with the same buffer at 0 C and eluted with water at room temperature. The eluted DNA was precipitated with ethanol and dissolved in 10 mM Tris.HCl pH 7.3 with 1 mM EDTA.

The oligo (dG) tailed linker DNA was prepared by digesting 75 pg of pLl DNA with 20 U of PstI endonuclease in 450 Ul containing 6 mM Tris.HCl pH 7.4, 6 mM MgCl 2 6 mM 2-ME, 50 mM NaC1, and 0.01 mg of BSA per ml. After 16 hr at 30 0 C the reaction mixture was extracted with phenol-CHC1 3 i and the DNA was precipitated with alcohol. Tails of 10 to 15 deoxyguanylate (dG) residues were then added per end with 46 U of terminal deoxynucleotidyl transferase in the same reaction mixture (38 4l) as described above, except that 0.1 mM dGTP replaced dTTP. After 20 min. at 37 0 C the mixture was extracted with phenol-CHCl 3 and after the DNA Clrrrpl=~ C ~IC3---C P-~ t was precipitated with ethanol it was digested with U of HindIII endonuclease in 50 ul containing mM Tris.HCl pH 7.4, 7 mM MgC1 2 60 mM NaC1, and 0.1 mg of BSA at 37 0 C for 4 hr. The small oligo (dG)-tailed linker DNA was purified by agarose gel electrophoresis and recovered as described above.

cDNA Library Preparation: Step 1: cDNA synthesis. The reaction mixture j (10 pl) contained 50 mM Tris.HCl pH 8.3, 8 mM MgCl 2 mM KC1, 0.3 mM dithiothreitol, 2 mM each dATP, dTTP, dGTP, and dCTP, 20 pCi 3 2 P-dCTP (3000 Ci/mmole), 6 pg polyA RNA from Con-A induced LB2-1, 60 units RNasin (Biotec, Inc., Madison, WI), and 2 ug of the vectorprimer DNA (15 pmol of primer end), and 45 U of reverse transcriptase. The reaction was incubated 60 min at 42 0 C and then stopped by the addition of 1 ul of 0.25 M ETDA (pH 8.0) and 0.5 ul of 10% SDS; 40 ul of phenol- SCHC13 was added, and the solution was blended t vigorously in a Vortex mixer and then centrifuged.

After adding 40 il of 4 M ammonium acetate and 160 ul t of ethanol to the aqueous phase, the solution was chilled with dry ice for 15 min., warmed to room temperature with gentle shaking to dissolve unreacted deoxynucleoside triphosphates that had precipitated **during chilling, and centrifuged for 10 min. in an Eppendorf microfuge. The pellet was dissolved in 10 ul f i *of 10mM Tris.HCl pH 7.3 and 1 mM EDTA, mixed with 10 ul of 4 M ammonium acetate, and reprecipitated with 40 il Sof ethanol, a procedure which removes more than 99% of unreacted deoxynucleoside triphosphates. The pellet was rinsed with ethanol.

I Y I I -31a d i t I i I t t t t S I I I S«ft r~l Step 2: Oligodeoxycytidylate [oligo (dC)] addition. The pellet containing the plasmidcDNA:mRNA was dissolved in 20 pl of 140 mM sodium mM Tris.HCl pH 6.8 buffer containing 1 mM CoC12, 0.1 mM dithiothreitol, 0.2 ug of poly(A), 70 uM dCTP, 5 pCi 3 2 P-dCTP, 3000 Ci/mmole, and 60 U of terminal deoxynucleotidyl transferase. The reaction was carried out at 370C for 5 min. to permit the addition of 10 to 15 residues of dCMP per end and then terminated with 2 pl of 0.25 M EDTA (pH 8.0) and 1 pl of 10% SDS. After extraction with 20 pl of phenol- CHC1 3 the aqueous phase was mixed with 20 pl of 4 M ammonium acetate, the DNA was precipitated and reprecipitated with 80 ul of ethanol, and the final pellet was rinsed with ethanol.

Step 3: HindIII endonuclease digestion. The pellet was dissolved in 30 pi of buffer containing mM Tris.HCl pH 7.4, 7 mM MgCl 2 60 mM NaC1, and 0.1 mg of BSA per ml and then digested with 10 U of HindIII endonuclease for 2 hr at 370C. The reaction was terminated with 3 ul of 0.25 M EDTA (pH 8.0) and 1.5 1 of 10% SDS and, after extraction with phenol-CHC1 3 followed by the addition of 30 pl of 4 M ammonium acetate, the DNA was precipitated with 120 pl of ethanol. The pellet was rinsed with ethanol and then dissolved in 10 pl of 10 mM Tris.HCl (pH 7.3) and 1 mM EDTA, and 3 pl of ethanol was added to prevent freezing during storage at -200C.

Step 4: Cyclization mediated by the oligo (dG)tailed linker DNA. A 9 Vi sample of the HindIII endonuclease-digested oligo (dC)-tailed cDNA:mRNA plasmid (about 90% of the sample) was incubated in a mixture (90 pi) containing 10 mM Tris.HC1 pH 7.5, 1 mM EDTA, 0.1 M NaCI, and 1.8 pmol of the oligo (dG)-tailed 41.

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I IE reI 4o 4 I 44: 4 4 4 44a linker DNA at 65 0 C for 5 min, shifted to 42 0 C for min, and then cooled to 0 C. The mixture (90 Pl) was adjusted to a volume of 900 pl containing 20 mM Tris.HCl pH 7.5, 4 mM MgC12 10 mM (NH 4 2

SO

4 0.1 M KC1, pg of BSA per ml, and 0.1 mM B-NAD; 6pg of E. coli DNA ligase were added and the solution was then incubated overnight at 12 0

C.

Step 5: Replacement of RNA strand by DNA. To replace the RNA strand of the insert, the ligation mixture was adjusted to contain 40 IiM of each of the four deoxynucleoside triphosphates, 0.15 mM 8-NAD, 4 pg of additional E. coli DNA ligase, 16 U of E. coli DNA polymerase I (Poll,) and 9 U of E. coli RNase H. This mixture (960 ul) was incubated successively at 12 0 C and room temperature for 1 hr each to promote optimal repair synthesis and nick translation by Poll.

Step 6: Transformation of E. coli.

Transformation was carried out using minor modifications of the procedure described by Cohen et al. (Proc. Nat. Acad. Sci. 69: 2110-2114 [1972]). E. coli K-12 strain MC1061 (Casadaban, M. and Cohen, J. Mol. Biol. 138: 179-207 [19801) was grown to 0.5 absorbancy unit at 600 nm at 37 0 C in 20 ml of L-broth. The cells were collected by centrifugation, suspended in 10 ml of 10 mM Tris.HCl pH 7.3 containing 50 mM CaCl2, and centrifuged at 0 C for min. The cells were resuspended in 2 ml of the above buffer and incubated again at 0 C for 5 min.; then, 0.2 ml of the cell suspensions was mixed with 0.1 ml of the DNA solution (step 5) and incubated at 0 C for min. Next the cells were kept at 37°C for 2 min. and thereafter at room temperature for 10 min.; then 0.5 ml of L-broth was added, and the culture was in%:ubated at 37 0 C for 30 min., mixed with 2.5 ml of L-broth soft t t 4 44 4 t 4 4 i -33agar at 42 0 C, and spread over L-broth agar containing ug of ampicillin per ml. After incubation at 37 0

C

for 12 to 24 hr, individual colonies were picked with sterile tooth-picks. In all, approximately 1 x 105 independent cDNA clones were generated.

G. Size Fractionated Sub-library pg of plasmid DNA representing the entire cDNA library (pcD-X DNA) was digested separately with the restriction enzymes SalI, HindIII, and Clal to linearize the plasmid. The restricted DNAs were siz, fractionated on a 1% agarose gel to separate plasmids having different size cDNA inserts. Segments were excised from the gel representing plasmids with cDNA inserts of the following size ranges: 0 -lkb 1 -2kb 1 2 -3kb 3 -4kb i 4 5 kb and longer.

DNA was eluted from each gel slice using the glass i i powder method of Vogelstein and Gillespie (Proc. Nat.

Acad. Sci. 76: 615-619 [19701). The eluted ii c DNAs from the 3 digests were pooled on the basis of 0 t4t 1size, and treated with T4 ligase to recyclize in a I total volume of 15 pil containing 50 mM Tris.HCl pH 7.4, mM MgC1 2 10 mM DTTI 1 mM spermidine, 1 mM ATP and S100 ug/ml BSA. The ligation reactions were incubated 16 hr at 12 0 C. About 3 il of each combined size fraction was used to transform E. coli strain MC 1061 using the method of Cohen, S. et al. (Proc. Nat. Acad.

i -i i -34- Sci. 69: 2110-2114 [1972]). A library of about 105 independent transformations was obtained for the fraction containing cDNA inserts 1-2 kb in 15 length.

A collection of 104 independent clones were picked at random from the sublibrary enriched for cDNA inserts of 1-2 kb and propagated individually in wells of microtiter dishes containing 200 Wl L broth with ampicillin at 50 pg/ml and dimethyl sulfoxide at 7%.

H. Screening of 1-2 kb Sub-library by DNA Transfections Pools containing 48 cDNA clones were prepared from the microtiter cultures. 58 such pools were grown up in 1 liter cultures of L-broth containing 100 ug/ml ampicillin. Plasmid DNA was isolated from each culture and purified by twice banding through CsCl gradients.

The DNA representing each pool was transfected into COS-7 monkey cells as follows.

One day prior to transfection, approximately 106 COS-7 monkey cells were seeded onto individual 60 mm plates in DME containing 10% fetal calf serum and 2 M glutamine. To perform the transfection, the medium was aspirated from each plate and replaced with 1.5 ml of DME containing 50 mM Tris.HCl pH 7.4, 400 ug/ml DEAE- Dextran and 15 ug of the plasmid DNAs to be tested.

The plates were incubated for four hours at 37 0 C, then the DNA-containing medium was removed, and the plates were washed twice with 2 ml of serum-free DME. DME containing 150 pM chloroquine was added back to the plates which were then incubated for an additional 3 hrs at 37 0 C. The plates were washed once with DME, and then DME containing 4% fetal calf serum, 2 mM glutamine, penicillin and streptomycin was added. The cells were then incubated for 72 hrs at 37 0 C. The I 44 4* 4t 41 4 1 *4 444 I 44 4 4 (1 4444 1 4 444 i L i tIt St I Sl

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growth medium was collected and assayed for murine IL-2 activity as described above.

Four pools (groups 6, 33, 40, and 56) yielded murine IL-2 activity (see Table II below). Group 33 was then subdivided into 8 pools, each containing 6 of the original pools. Only one of these subpools (group c) was positive in the transfection assay. Each of the plasmids in group c was transfected individually into COS-7 cells. Only one clone, designated MT-18, was active in producing TCGF activity.

Restriction mapping of the MT-18 cDNA clone identified a PstI-HindIII fragment within the cDNA insert, as shown in Figure 2A. This fragment was purified by electrophoresis through a 5% poly acrylamide gel, and eluted. This fragment was labelled with 3 2 P-dCTP by nick translation according to Maniatis, T. et al. ("Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, U.S.A. [1982]).

The pools of 48 cDNA clones were transformed from the tiicrotiter trays to nitrocellulose filters placed on plates of L-agar 100 pg/ml ampicillin. These plates were incubated overnight at 37 0 C. The bacterial colonies were lysed and the DNA bound to the filters as described in Maniatis, et al. (above).

The nick-translaled PstI-HindIII fragment from MT- 18 was hybridized with each of the 58 filters representing the pools. The hybridizations were performed in 6XSSPE (0.18 M NaCi, ImM EDTA, and 10 mM NaH 2

PO

4 [pH 7.91), 50% formamide, 100 Ug/ml E. coli tRNA, 0.1% SDS, at 42 0 C overnirht. The filters were washed once at room temperature in 2X SSPE 0.1% SDS, and then twice with 0.2X SSPE at 60 0 C. Following incubation of the filters, 15 individual clones were found to hybridize with the probe.

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-36- Plasmid DNA from each of these clones was prepared as described above, and transfected into COS-7 cells.

Of these clones, only three, contained in pools 6 (MT- 40 (MT-20), and 56 (MT-28) produced high levels of muriLne IL-2 activity (see Table II). Restriction analysis showed that the clones share essentially the same structure.

f f, -37- TABLE II DNA Transfection Assay for Murine IL-2 Activity From Pools of Plasmid DNA DNA units/mi DNA units/mi DNA units/mi First screening 4 4. 4 4, I I *44 4 .4 4$ 4 4 44 4 4 4 4 44 4444 4 4444 4 44 4 4*4 4 44 44 4 4 *4 4444 I I 4444

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TABLE H1 (cont'd.) DNA units/mi ENA units/mi DNA units/ml Third screening: single clones fran group C Fourth screening: single clones hybridized with cDNA insert of clone MT-18 1 2 3 (MT-18) MTr-1 MT-2 MT-3 MT-4 <10 <10 9020 5851 <10 <10 <10 <10 4 6 MT-7 MT-17 MT-1 9 <10 <10 <10 6800 <10 MT-22 MT-23 MT-2 6 MT-28 Kf-2 9 8200 41 4, Ill 4 I It I I 4 tat x at 14 Il 4 I I 4 II #4 1 4 'I

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4*4,44 4 Mock-infected CcS-7 II -39- A plasmid (pcD-IL-2) carrying a substantially full-length cDNA insert is shown in Figure 2, and an E.

coli bacterium (MC1061) carrying the plasmid has been deposited with the ATCC (accession number 39892). In Figure 2, transcription of the 816 bp cDNA insert contained in the pcD expression vector from the early promoter is indicated by the arrow, The locations of the splice donor and acceptor sites are shown. The polyadenylation signal, also derived from SV40, is located at the 3'-end of the cDNA insert. The IL-2 coding region in the cDNA insert is heavily shaded while the non-coding regions are lightly shaded. The remainder of the vector sequences are derived from pBR322, including the B-lactamase gene (Amp

R

and the origin of replication.

io Utilizing both the M13 dideoxy chain Stermination method (Sanger, et al., Proc. Natl. Acad.

Sci. U.S.A. 74: 5463-5467 [1977]) and modified t Maxam/Gilbert technique (Rubin, C. and Schmid, C., Nucleic Acid Res. 8: 4613-4619 [1981]) the MT-1 sequence was determined. The cDNA insert contains a single open reading frame. The first ATG is found 49-51 nucleotides froin the 5' end, and is followed by 168 codons before the termination riplet at nu'leotide positions 553-555.

Figure 3 is a comparison between the putative mouse and human IL-2 protein sequences. Identical residues (after S,4* alignment) are shown in shaded boxes. Surprisingly, 89 r of the predicted mouse IL-2 rtsidues are conserved in r human IL-2.

The homologies between the mouse IL-2 cDNA and human IL-2 cDNA are also surprising. Overall, there is about 70% homology between the two IL-2 cDNA sequences (Brutlag, D. et al., Nucleic Acids Res. 10: 279-294 [1981]). In particular, the regions covered by I 1~11~~ nucleotide positions 1-125, 229-354, and 416-680 share extensive homology with the corresponding regions of human IL-2 cDNA. However, the trinucleotide CAG sequence, which is repeated twelve times within the mouse IL-2 cDNA coding region, is not present in any human IL-2 cDNA isolated to date.

The clone library in the pcD expression vector enabled the identification of complete cDNA clones by direct expression in mammalian cells. Specifically, complete mouse IL-2 cDNA clones were directly identified by transfecting COS-7 cells with randomly picked cDNA clones, and measuring the TCGF activity secreted into the i cell supernatant. These results indicate that the i identification of full-length cDNA clones of lymphokines or hormones may be achieved solely on the basis of i detection of a functional polypeptide in eukaryotic cells. Identification of relevant cDNA clones based on the functional expression of the gene offers advantages to hybrid selection procedures.

Identification of cDNA clones by hybrid selection relies on nucleotide sequence homology between SmRNA and cDNA inserts, which can prove misleading. In fact, some of the functionally inactive cDNA clones which hybridized with the mouse IL-2 cDNA probe (MT-3p MT-4, "t °MT-5, MT-7, MT-17, MT-19, MT-21, MT-22, MT-23, and MT-29 in Table II) have different restriction maps from that of j 'the functional mouse IL-2* cDNA. Interestingly, these 4 inactive clones also hybridized with a human IL-2 cDNA Sprobe. Thus, it is possible to isolate irrelevant clones on the basis of partial sequence homology among the cDNA inserts. Furthermore, the cloning protocol disclosed herein is particularly useful in cases where the large amounts of mRNA needed for hybrid selection are not available.

-41- The invention further provides a method of identifying individual nucleotide sequences coding for a polypeptide, said method comprising the stepsof: a) locating a cellular source expressing the polypeptide; b) constructing from the cellular source a cDNA library composed of vectors, each vector containing a cDNA insert positioned to permit expression of the insert after incorporation into a eukaryotic cell; c) screening the library by tr.ansfecting or transforming eukaryotic cells with the vectors and assaying the eukaryotic cells for the presence of the rolypeptide; and d) isolating the vector containing the cDNA insert encoding the polypeptide.

ij From the foregoing, it will be appreciated that the cDNA clones of the present invention provide accurate R and complete sequence data on murine IL-2. The invention j also provides to those skilled in the art means for producing significant quantities of the factor (essentially free from other hematopoietic factor) for «the improved in vitro maintenance of T cells and other hematopoietic cells. Further, the information gleaned from the cDNA clones increases understandi:" of the i mammclian immune response, enhancing experimental Si research capabilities.

Claims (20)

1. A process for producing a polypeptide exhibiting murine interleukin-2 activity and having the structure: C rt C t t Cf C Met Tyr Ser Met Leu Val Leu Leu Thr Ser Ser Ser Gin Gin Gin Gin Met Asp Leu Gin Arg Asn Leu Lys Tyr Leu Pro Lys Cys Leu Glu Asp Asp Leu Thr Gin Glu Asn Phe Ile Leu Lys Gly Ser Asp Glu Ser Ala Ile Ala Phe Cys Gin Leu Val Asn Thr Ala Gin Gin Glu Leu Leu Pro Gin Ala Glu Leu Ser Lys Ser Asn Asp Asn Thr Val Gin Ser Ala Ser Glu Gln Leu Arg Thr Gly Ser I!,e Thr Val Ile Ser Ala Ala His Ser Met Glu Pro Phe Arg Phe Asp Ile Cys Val Pro Thr Gln Gin Leu Glu Arg Met Leu Thr Leu Lys Leu Arg Gin Leu Val Thr Glu Cys Phe Leu Ser Thr Thr Ser Gin Gin Glu Phe Asp His Glu Val Gin Arg Ser Leu Thr Ser Ser Gin Gin Leu Leu Asn Tyr Lys Phe Leu Gin Val Leu Asp Ala Val Lys Phe Asp Arg Trp Pro Gin, L ~-i Li1- said process comprising the steps of: a) providing a vector comprising a nucleotide sequence coding for said polypeptide, wherein the nucleotide sequence is capable of being expressed by a host containing the vector and is a cDNA sequence derived from an mRNA sequence coding for said polypeptide; b) incorporating the vector into the host; and c) maintaining the host containing the vector under conditions suitable for expression of the nucleotide sequence into said polypeptide. I -43-

2. A process as claimed in claim 1 wherein the vector comprises the nucleotide sequence coding for said polypeptide linked to a second nucleotide sequence, and the second nucleotide sequence comprises a promoter sequence which promotes expression of the nucleotide sequence coding for said polypeptide.

3. A process as claimed in claim 1 or claim 2 wherein the host is a mammalian cell transformed or transfected with the vector, which includes a promoter sequence for the nucleotide sequence, together with a polyadenylation sequence.

4. A process as claimed in claim 3 wherein the second nucleotide sequence comprises an SV40 virus early Sregion promoter and an SV40 virus late region polyadenylation sequence. A process as claimed in any of claims 1 to 4 wherein the nucleotide sequence includes a portion coding for at least a part of a signal sequence of the polypeptide. j 6. A recombinant polypeptide consisting essentially of the amino acid sequence defined in claim 1 and exhibiting murine interleukin-2 activity.

7. A polypeptide as claimed in claim 6 without a Jj signal sequence.

8. A polypeptide as claimed in claim 6 in substantially pure form and essentially free from other mammalian hematopoietic cell proteins. 'NT j 1_ -44-

9. cDNA clones coding for the polypeptide having the sequence given in claim 1. 0. A nucleic acid molecule comprising acDNA 10'ef'ev 7 a1r ikA? 7A SeL/etce sequence 4 coding for the polypeptide defined in claim 1.

11. A vector consisting essentially of the DNA sequence of claim

12. A replicable vector including and capable of expressing the DNA sequence of claim 10, when said vector is incorporated into a microorganism or cell.

13. A microorganism or cell transformed or transfected with the replicable expression vector of claim 12.

14. A process for enhancing T cell growth in vivo in a non-human animal, comprising contacting the cell with a polypeptide having the amino acid sequence defined in claim 1, or said sequence without its leader sequence. Ij 15. A process for enhancing T cell growth in vitro EI comprising contacting the cell with a polypeptide having S the amino acid sequence defined in claim 1, or said sequence without its leader sequence.

16. A process for preparing the polypeptide set forth in claim 1, which comprises cultivating, in an *aqueous nutrient medium, a suitable prokaryotic microorganism or eukaryotic cell which has been transfected or transformed with a vector comprising a sufficient portion of the cDNA sequence shown in Figure 1 to encode said polypeptide. i 45

17. An organism or cell transformed with a cDNA sequence derived from an mRNA sequence coding for the polypeptide having the amino acid sequence defined in claim 1.

18. The protein shown in claim 1 whenever produced by cultivating the organism or cell of claim 17.

19. A recombinant DNA molecule consisting of segments of DNA derived from different genomes which have been joined end to end outside of living cells and have the capacity to infect some host and to be maintained therein, and the progeny thereof, wherein one DNA segment is a cDNA sequence derived from an mRNA sequence which encodes the polypeptide of claim 1. Recombinant murine interleukin-2 having the amino acid sequence given in claim 1.

21. Recombinant murine interleukin-2 as claimed in claim 20, substantially free of other murine proteins.

22. A process for producing a polypeptide substantially as described herein and with reference to the Examples and Drawings.

23. A recombinant polypeptide substantially as described herein and with reference to the Examples and Drawings.

24. A process for enhancing T-cell growth in vivo substantially as described herein and with reference to the Examples and Drawings. I 25. A process for enhancing T-cell growth in Aitro substantially as described herein and with reference to the Examples and Drawings. c I DATED this 4th day of May 1990 SCHERING BIOTECH CORPORATION By their Patent Attorneys GRIFFITH HACK CO 7 7S/sy 48 355/185 1020 30 .40 TATCACCCTT GCTAATCACT CCTC'ZAGTG ACCTCAAGTC 63 CTGCAGGC ATG TAC AGC A7G CAG .MET Tyr Ser M'ET Gin y qo 00 0 000 4 0 OS 0 0 5 000 0 00 0$ 01 t 0 0 I I' CTC Lou TCA Ser CAG Gin 228 AGC Ser TIT Phe GAA Glu TTG Leu MAG Lys 498 GTG Val GCA TCC TGT Ala Her Cys 123 AGC TCC ACT Ser Bar Thr 183 CAG CAG CAG Gin Gin Gin AGG ATG GAG Arg MET Glu 288 TAC TTG CCC Tlyr Leu Pro ICTT GGA CCT LeU Gly Pro 393 GMA GAT GCT Giu Amp Ala 453 GGC TCT GAC Gly Ser Asp GAC TTT CTG Asp Phe Leu 710 LOU 138 TCT Ser CTG Lou AGG Arg GCC Ala CAT H is 408 TTC Phe TIT Phe TGG CTT Lou GCG Ala 198 CAG Gin CTG Lou GMA Giu CTG Lou AGC Ser 468 TGC Cy a GCC 93 108 (j7G CTC CTT GTC AAC AGC GCA CCC ACT Val Lou Lou Val Amn Ser Ala Pro Thr 153 168 GMA GCA CAG CAG CAG CAG CAG CAG CAG Glu Ala Gin Gin Gin Gin Gin Gin Gin 213 CTG 'rIG ATG GAC CTA CAG GAG CTC CTG Lou Lou MET Asp Lou Gin Giu Lou Lou ,258 273 AM CTC CCC AGG ATG CTC ACC TTC MAA Lys Lou Pro Arg MET Lou Thr Phe Lys '318 333 'IG MAA OAT CTT CAG TGC' CTA GMA GAT Lou Lys Asp Lou Gin Cys Lou Glu Asp 363 378 GAT TIC ACT CAM AGC AMA AGC TTT CAA Asp Lou Thr Gin Ser Lys Her Phe Gin 423 438 AMT ATC AGA GTA ACT GTT GTA MAA CTA Asn Ile Arg Val Thr Val Val Lys Leu 483 CMTIC GAT OAT GAG TCA GCA ACT GTG Gin 5 Phe Asp Asp Giu Ser Ala Thr Val 528 543 TIC TOT CMA AGC ATC ATC TCA ACA AGC Ph. Cys Gin Ser Ile Ile Sor Thr Her 590 600 610 ACATAAGZCT CTCTATTTAT TTAAATATTT 650 660 670 680 rATCTTTT GTAMCTACTA GTCTTCAGAT GATAM IZATG 720 730 740 750 ;GCTCAAA AMTGTTTTAA ACtATTTATC TGAAATTATT 790 800 810 820 3TAGACTC ATTAATAAMA GTATTAGAT GATTCAAATA C C Ct 510* 4 0 .140.. 6 440* 0000 Arg Trp Ile Ala 570 580 TAACT ATGTACCTCC TGCTTACMAC CCT CMA Pro Gin I L 4 620 630 640 AACTTTAATT TATTTTTGGA TGTATTGTTT AC! 690 700 710 GATCTT-TAAA GATTCTTTTT GTMAGCCCCA AGM 760 770 780 TATTATATTG A&TTarTAAA TATCATGTGT AG4 TAAAAA Figure I 48 355/85 Hind lI SV4Oori AmpR splice junction ',Stl G-C tail k-PstI PstI pBR322 oDNA insert Hind JIE polyA 6-C .JTail 4 Hind IL flv;I]r TaqI A-T Tail PstI PV*UIE HaeILHindMff AcicI Figure 2 201 Mouse MYS A V V LVSPSSS SSSTAEAQQQQQQQQ Human S 26t s0 Mouse QQQHJQ L MffC.. ELfSRME:R :Lrn PRMLTFKFY LMKo ATE Human L H IL.L1I~ MIJILNG IN~lX K ~!P1kT RML FkFY m'PK Kkd~ 100

120. Mose LR 1 CLD GLHVDLT QSKS soLEDAENF[i IM1R ~T V Human L!kH LOCLEE1~~ E LEJKNAJS RPRDL- ISIN I JL 100 140 160 169 Mouse K LGDNTE 6 QFDbE- s T LRRW. ~S A[l iSPQ Human GI TEFMEY dTfA F .R T[FC. IIS LT 120 140 153 co Figure 3 M1 I 48 355/85 A Leader Sequence Is pt IsI "ar a-factor Lys Arg G~uAidM Glu Ais Trp Ke Trp Lou Gin Lou Lys PIro Gty B HIndX3 aIs aS cx spacer Gin Pro WIt Tyr Lys Aig Gki Ale StuI XhoI V. ,;~ATG -AAA AGA GAG GCT- 2-et-Lys Arg Giu Ala- IR veL *#04 1111 -AAA AGCTT- AAA AGGI CC ITCGAGG RI Pv1L 2pum R1 od ARSI Sam, pTRP584 Figure 4